1. Field of the Invention
The present invention relates generally to the fields of microbiology, molecular biology and virology. More particularly, it concerns methods of generating infectious reovirus containing selected modifications using cloned DNA.
2. Description of Related Art
Mammalian orthoreoviruses (reoviruses) are members of the Reoviridae family. Reoviruses contain 10 double-stranded (ds) RNA gene segments enclosed in two concentric protein shells, outer capsid and core (Nibert and Schiff, 2001). These viruses serve as a versatile experimental system for studies of viral replication events at the virus-cell interface, including engagement of cell-surface receptors (Barton et al., 2001a), internalization and disassembly (Ebert et al., 2002; Ehrlich et al., 2004), and activation of the innate immune response, including NF-κB-dependent cellular signaling pathways (Connolly et al., 2000; O'Donnell et al., 2006). Reoviruses also provide a model system for studies of virus-induced apoptosis and organ-specific disease in vivo (O'Donnell et al., 2005).
With the exception of dsRNA viruses, a reverse genetics system exists for all major groups of animal RNA viruses, including picornaviruses (Racaniello and Baltimore, 1981), rhabdoviruses (Schnell et al., 1994; Lawson et al., 1995; Whelan et al., 1995) paramyxoviruses (Collins et al., 1995; Garcin et al., 1995; Yoneda et al., 2006), bornaviruses (Schneider et al., 2005), flaviviruses (Rice et al., 1989; Gritsun and Gould, 1995; Kinney et al., 1997; Yun et al., 2003), bunyaviruses (Bridgen and Elliott, 1996), orthomyxoviruses (Fodor et al., 1999; Neumann et al., 1999), and coronaviruses (Almazan et al., 2000; Yount et al., 2003; Coley et al., 2005). Notably, viral genome structure does not appear to dictate the potential for plasmid-based virus recovery since monopartite and segmented RNA viruses of both positive and negative polarity have been generated by reverse genetics methods. As positive-strand virus genomic RNA is competent for translation, productive viral infections have been initiated by transfection of cells with plasmids that express full-length viral mRNA or, more commonly, with viral RNA transcribed in vitro from cDNA templates. In contrast, genomic RNA of negative-strand viruses is incapable of autonomous replication and requires coexpression of viral replication proteins to reconstitute functional replication complexes on full-length viral antigenomic or genomic RNA transcribed from transfected plasmids by bacteriophage or cellular RNA polymerase.
Despite extensive efforts in several laboratories, generation of an animal dsRNA virus entirely from cloned cDNAs has not been achieved. This critical technological gap is perhaps the single most important limitation to studies of these viruses. Previous work on reovirus and rotavirus reverse genetics has resulted in entirely RNA-based (Roner et al., 1997) or partially plasmid-based (Komoto et al., 2006) systems that permit replacement of one or two viral genes. These approaches have been used to rescue temperature-sensitive reovirus strains (Roner et al., 1997), define packaging signals in reovirus RNAs (Roner and Steele, 2006), and isolate rotaviruses containing engineered changes in the viral attachment protein (Komoto et al., 2006). However, neither the reovirus nor rotavirus reverse genetics systems in their current configurations permit selective introduction and recovery of desired mutations in each viral gene segment.